Effect of berberine on the proliferation and apoptosis of lung cancer stem cells and the possible mechanism

doi:10.3969/j.issn.2095-4344.2017.09.001

Abstract

Abstract:

BACKGROUND: Previous studies have demonstrated that berberine represses multiple tumors and tumor stem cells, but the effect of berberine on lung cancer stem cells (LCSCs) remains unclear.
OBJECTIVE: To explore the effect of berberine on the proliferation and apoptosis of LCSCs and the possible mechanism.
METHODS: CD133⁺ LCSCs were separated from A549 cells by immunomagnetic beads. The effects of different
concentrations (0, 2.5, 5, 10, 20, 40 mg/L) of berberine on the proliferation and apoptosis of LCSCs were determined by MTT and flow cytometry analysis, respectively. In order to further affirm the effect of berberine on the proliferation and apoptosis of LCSCs, the expression levels of Ki67, Bax and Bcl-2 protein were detected by western blot. In addition, to investigate the potential mechanism by which berberine exerts regulatory effects on LCSCs, the expression levels of Hedgehog signaling pathway-associated proteins (PTCH1, SHH, Gli-1 and SMO) were determined.
RESULTS AND CONCLUSION: After magnetic cell sorting, the content of the CD133⁺ fraction was enriched up to 84.13%. MTT and flow cytometry assays showed that berberine inhibited proliferation and promoted apoptosis of LCSCs in a concentration-dependent manner. Western blot analysis showed that the expression levels of Ki67, Bcl-2, PTCH1, SHH, Gli-1 and SMO proteins of LCSCs cultured in the medium with 20 mg/L berberine were dramatically decreased compared to the control, while the expression level of Bax protein was markedly increased compared to the control. These findings suggest that berberine may inhibit proliferation and promote apoptosis for LCSCs through the Hedgehog signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lung Neoplasms, Neoplastic Stem Cells, Berberine, Hedgehog Proteins, Tissue Engineering

CLC Number:

R394.2

Sun Yan-zhen, Li Zhen, Yuan Zheng. Effect of berberine on the proliferation and apoptosis of lung cancer stem cells and the possible mechanism[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1313-1318.

Figures/Tables 2

2.2 小檗碱抑制肺癌干细胞增殖为了探究小檗碱对肺癌干细胞增殖的影响，将处于对数生长期的肺癌干细胞在含有不同质量浓度小檗碱的培养液中培养24 h，采用MTT法检测小檗碱对肺癌干细胞增殖的影响。结果发现含有2.5，5，10，20，40 mg/L小檗碱的培养液培养的活细胞分别为未加药组的78.5%，63.4%，52.1%，43.9%和40.7%，可见小檗碱能够抑制肺癌干细胞生长，并呈浓度依赖性。采用免疫印迹检测未加药组和20 mg/L小檗碱组的增殖相关蛋白Ki67的表达水平，发现20 mg/L小檗碱组的Ki67表达水平显著低于未加药组，这也表明了小檗碱能够抑制肺癌干细胞增殖(P < 0.05，P < 0.01，P < 0.001，图2)。 2.3 小檗碱促进肺癌干细胞凋亡为了探究小檗碱对肺癌干细胞凋亡的影响，将处于对数生长期的肺癌干细胞在含有不同质量浓度小檗碱(0，2.5，5，10，20，40 mg/L)的培养液中培养24 h，用流式细胞仪检测其凋亡率，各组的凋亡率分别为0.81%，7.21%，15.8%，28.2%，38.7%，40.5%，可见小檗碱能够促进肺癌干细胞凋亡，并呈浓度依赖性。采用免疫印迹检测未加药组和20 mg/L小檗碱组的凋亡相关蛋白Bax和Bcl-2的表达水平。结果发现20 mg/L小檗碱组细胞中Bax蛋白的表达水平显著高于未加药组，而Bcl-2蛋白的表达水平显著低于未加药组，这也表明了小檗碱能够促进肺癌干细胞凋亡(P < 0.05，P < 0.01，P < 0.001，图3)。 2.4 小檗碱抑制肺癌干细胞中Hedgehog信号通路的活性为了探究小檗碱是否通过调控Hedgehog信号通路对肺癌干细胞的增殖和凋亡起作用，将处于生长对数期的肺癌干细胞培养在未加药和加有20 mg/L小檗碱的培养液中培养24 h，检测两组细胞中Hedgehog信号通路中PTCH1、SHH、Gli-1和SMO蛋白的表达。结果发现这4种蛋白在加药组细胞中的表达水平明显低于未加药组，这表明小檗碱抑制了Hedgehog信号通路的活性(P < 0.05，P < 0.01，图4)。所以，作者推测小檗碱对肺癌干细胞增殖和凋亡的影响可能是通过调控Hedgehog信号通路起作用。"

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